The accumulation of viscous mucus in the airways is the primary cause of long-term bacterial infections, respiratory failure, and eventually death in cystic fibrosis (CF). The inflammatory response to infections in the airways leads to the pathological release of cytoskeletal proteins, DNA and other polyelectrolytes, which cause the electrostatic assembly of large aggregates stabilized by cationic ligands in CF mucus, and results in the sequestration of endogenous antibacterial polypeptides and contributes to the loss of antimicrobial function. The long-range goal of this proposal is the rational design of therapeutic strategies in the restoration of antimicrobial function in CF, based on a biophysical understanding of electrostatic interactions in CF mucus. The three specific aims of the proposal are: (1) To determine the ionic strength within the airway surface liquid (ASL) expressed on cultured human airway epithelial cell lines with and without a functioning cystic fibrosis transmembrane conductance regulator (CFTR). (2) To characterize quantitatively the structural form and thermodynamic stability of electrostatic complexes of endogenous antibacterial peptides and anionic polyelectrolytes in the ASL under CF physiological conditions, and design biophysical therapeutic strategies to unbind these sequestered antibacterial peptides. (3) To develop, via directed molecular evolution, an anionic DNAzyme which mimics the biofilm suppression action of cationic lactoferrin, but will not be sequestered due to its opposite charge.